It is now aparent that regulated exocytosis is not restricted to specialized secretory cells, and that conventional lysosomes can respond to elevations in intracellular free Ca2+ by fusing with the plasma membrane. Investigating the physiological function of this ubiquitous lysosomal exocytic pathway, we found that it plays a central role in the mechanism by which mammalian cells reseal their plasma membrane after injury. Our previous studies also implicated synaptotagmin VII (Syt VII), a widely expressed member of the synaptotagmin family of putative Ca2+ sensors, in the regulation of lysosomal exocytosis and plasma membrane resealing. We now plan to expand our studies of the physiological role of Ca2+-regulated lysosomal exocytosis, and to characterize unique molecular interactions and dynamic propel-ties involved in this process. Our specific aims are: 1) To examine the resealing capacity of primary skin fibroblasts with genetic defects affecting lysosomes, using a collagen matrix contraction model of plasma membrane wounding and repair. Completion of this aim will allow us to determine if defects in plasma membrane resealing are a component of the serious symptoms associated with a series of lysosome-related genetic syndromes. 2) To investigate the role of the unconventional SNARE VAMP7 and of the synaptotagmin isoform Syt VII in the regulation of lysosomal exocytosis and plasma membrane repair. These studies will determine if Syt VII plays an essential role in the formation of specific SNARE complexes involved in lysosomal exocytosis, and will clarify the function of the lysosomal SNARE TIVAMP/VAMP-7, previously implicated in exocytic events associated with neurite outgrowth. 3) To verify if molecular interactions that regulate Ca2+-dependent exocytosis of conventional lysosomes also control secretion of the lysosome-related secretory granules of cytotoxic T lymphocytes (CTLs) and mast cells. These studies will provide important new information on the mechanisms regulating the exocytosis of lysosome-related granules that play critical roles in the immune response. 4) To investigate the spatial-temporal dynamics of Ca2+-regulated lysosomal exocytosis by total internal reflection fluorescence microscopy (TIR-FM). This powerful imaging technique will allow us to obtain quantitative parameters on the motion and distribution of lysosomes near the plasma membrane, map fusion sites on individual cells, and determine the diffusion coefficient of lysosomal membrane proteins after fusion. Taken together, the information generated by this project will significantly improve our understanding of fundamental biological processes involving regulated exocytosis of lysosomes, including maintenance of plasma membrane integrity and degranulation in cells of the immune system.